Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.

When Medical News Comes from Press Releases-A Case Study of Pancreatic Cancer and Processed Meat.

The media have a key role in communicating advances in medicine to the general public, yet the accuracy of medical journalism is an under-researched area. This project adapted an established monitoring instrument to analyse all identified news reports (n = 312) on a single medical research paper: a meta-analysis published in the British Journal of Cancer which showed a modest link between processed meat consumption and pancreatic cancer. Our most significant finding was that three sources (the journal press release, a story on the BBC News website and a story appearing on the 'NHS Choices' website) appeared to account for the content of over 85% of the news stories which covered the meta analysis, with many of them being verbatim or moderately edited copies and most not citing their source. The quality of these 3 primary sources varied from excellent (NHS Choices, 10 of 11 criteria addressed) to weak (journal press release, 5 of 11 criteria addressed), and this variance was reflected in the accuracy of stories derived from them. Some of the methods used in the original meta-analysis, and a proposed mechanistic explanation for the findings, were challenged in a subsequent commentary also published in the British Journal of Cancer, but this discourse was poorly reflected in the media coverage of the story.

Structural and functional characterization of a specific antidote for ticagrelor.

Ticagrelor is a direct-acting reversibly binding P2Y12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y12. Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 5'-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures.

Development of a homogeneous high-throughput screening assay for biological inhibitors of human rhinovirus infection.

Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule-1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti-ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.

The biology of protein kinase C.

This review gives a basic introduction to the biology of protein kinase C, one of the first calcium-dependent kinases to be discovered. We review the structure and function of protein kinase C, along with some of the substrates of individual isoforms. We then review strategies for inhibiting PKC in experimental systems and finally discuss the therapeutic potential of targeting PKC. Each aspect is covered in summary, with links to detailed resources where appropriate.

PKCepsilon regulates behavioral sensitivity, binding and tolerance to the CB1 receptor agonist WIN55,212-2.

The cannabinoid CB1 receptor (CB1) is one of the most abundant G protein-coupled receptors in the brain, but little is known about the mechanisms that modulate CB1 receptor signaling. Here, we show that inhibition or null mutation of the epsilon isozyme of protein kinase C (PKCepsilon) selectively enhances behavioral responses to the CB1 agonist WIN55,212-2 in mice, but not to the structurally unrelated CB1 agonist CP55,940. Binding affinity for [(3)H] WIN55,212-2 was increased in brain membranes from PKCepsilon(-/-) mice compared with PKCepsilon(+/+) mice. There was no difference in binding of the inverse agonist [(3)H] SR141716A. In addition, repeated administration of WIN55,212-2 produced greater analgesic and thermal tolerance in PKCvarepsilon(-/-) mice compared with PKCepsilon(+/+)mice. These results indicate that PKCvarepsilon selectively regulates behavioral sensitivity, CB1 receptor binding and tolerance to WIN55,212-2.

Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism.

Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.

A novel method for determination of the affinity of protein: protein interactions in homogeneous assays.

Nonradioactive homogeneous assays are widely used to screen for inhibitors of biomolecular interactions. To ensure optimal sensitivity for the detection of competitive inhibitors, reagent concentrations should be fixed at or below the K(D) of the protein-protein interaction. Accurate measurement of K(D) during assay development is therefore critical. Although conventional methods work well with heterogeneous assays, they are generally unsatisfactory with homogeneous systems. Here the authors describe an alternative method to determine the K(D) of protein-protein interactions in homogeneous assays. The method uses a rearrangement of the Cheng-Prusoff equation: IC50= (([Ki]/K(D)) x [L]) + Ki. A competitive inhibitor is titrated into the ligand-receptor binding assay at a range of ligand concentrations and IC50 values are calculated. Plotting measured IC50 versus concentration of ligand gives a linear plot with y-intercept (Ki) and gradient (Ki/K(D)). K(D) is the affinity constant for the ligand-receptor interaction. Here the authors use homogeneous time-resolved fluorescence (HTRF) in 2 model systems (TRAIL/TRAIL receptor 4 and OX40 ligand/OX40 receptor) and demonstrate that measured K(D) values calculated using the linearized Cheng-Prusoff plot compare favorably with those from independent experiments. The advantages and limitations of the method are discussed.

Protein kinase C epsilon regulates gamma-aminobutyrate type A receptor sensitivity to ethanol and benzodiazepines through phosphorylation of gamma2 subunits.

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Chronic ethanol exposure induces an N-type calcium channel splice variant with altered channel kinetics.

Chronic ethanol exposure increases the density of N-type calcium channels in brain. We report that ethanol increases levels of mRNA for a splice variant of the N channel specific subunit alpha1 2.2 that lacks exon 31a. Whole cell recordings demonstrated an increase in N-type current with a faster activation rate and a shift in activation to more negative potentials after chronic alcohol exposure, consistent with increased abundance of channels containing this variant. These results identify a novel mechanism whereby chronic ethanol exposure can increase neuronal excitability by altering levels of channel splice variants.